The present invention relates to recombinant DNA which encodes the BpmI restriction endonuclease as well as BpmI methyltransferase and expression of BpmI restriction endonuclease from E. coli cells containing the recombinant DNA. BpmI is an isoschizomer of GsuI (Fermentas 2000-2001 Catalog, Product No. ER0461/ER0462).
Type II restriction endonucleases are a class of enzymes that occur naturally in bacteria and in some viruses. When they are purified away from other bacterial proteins, restriction endonucleases can be used in the laboratory to cleave DNA molecules into small fragments for molecular cloning and gene characterization.
Restriction endonucleases act by recognizing and binding to particular sequences of nucleotides (the xe2x80x98recognition sequencexe2x80x99) along the DNA molecule. Once bound, they cleave the molecule within, to one side of, or to both sides of the recognition sequence. Different restriction endonucleases have affinity for different recognition sequences. Over two hundred and eleven restriction endonucleases with unique specificities have been identified among the many hundreds of bacterial species that have been examined to date (Roberts and Macelis, Nucl. Acids Res. 27:312-313 (1999)).
Restriction endonucleases typically are named according to the bacteria from which they are derived. Thus, the species Deinococcus radiophilus for example, produces three different restriction endonucleases, named DraI, DraII and DraIII. These enzymes recognize and cleave the sequences 5xe2x80x2TTT/AAA3xe2x80x2, 5xe2x80x2PuG/GNCCPy3xe2x80x2 and 5xe2x80x2CACNNN/GTG3xe2x80x2 respectively. Escherichia coli RY13, on the other hand, produces only one enzyme, EcoRI, which recognizes the sequence 5xe2x80x2G/AATTC3xe2x80x2.
A second component of bacterial restriction-modification (R-M) systems are the methyltransferases (methylases). These enzymes are complementary to restriction endonucleases and they provide the means by which bacteria are able to protect their own DNA and distinguish it from foreign, infecting DNA. Modification methylases recognize and bind to the same recognition sequence as the corresponding restriction endonuclease, but instead of cleaving the DNA, they chemically modify one particular nucleotide within the sequence by the addition of a methyl group (C5-methyl cytosine, N4-methyl cytosine, or N6 methyl adenine). Following methylation, the recognition sequence is no longer cleaved by the cognate restriction endonuclease. The DNA of a bacterial cell is always fully modified by the activity of its modification methylase. It is therefore completely insensitive to the presence of the endogenous restriction endonuclease. It is only unmodified, and therefore identifiably foreign DNA, that is sensitive to restriction endonuclease recognition and cleavage.
By means of recombinant DNA technology, it is now possible to clone genes and overproduce the enzymes in large quantities. The key to isolating clones of restriction endonuclease genes is to develop a simple and reliable method to identify such clones within complex genomic DNA libraries, i.e. populations of clones derived by xe2x80x98shotgunxe2x80x99 procedures, when they occur at frequencies as low as 10xe2x88x923 to 10xe2x88x924. Preferably, the method should be selective, such that the unwanted majority of clones are destroyed while the desirable rare clones survive.
A large number of type II restriction-modification systems have been cloned. The first cloning method used bacteriophage infection as a means of identifying or selecting restriction endonuclease clones (EcoRII: Kosykh et al., Mol. Gen. Genet. 178:717-719 (1980); HhaII: Mann et al., Gene 3:97-112 (1978); PstI: Walder et al., Proc. Nat. Acad. Sci. 78:1503-1507 (1981)). Since the presence of restriction-modification systems in bacteria enable them to resist infection by bacteriophage, cells that carry cloned restriction-modification genes can, in principle, be selectively isolated as survivors from genomic DNA libraries that have been exposed to phages. This method has been found, however, to have only limited value. Specifically, it has been found that cloned restriction-modification genes do not always manifest sufficient phage resistance to confer selective survival.
Another cloning approach involves transferring systems initially characterized as plasmid-borne into E. coli cloning plasmids (EcoRV: Bougueleret et al., Nucl. Acids. Res. 12:3659-3676 (1984); PaeR7: Gingeras and Brooks, Proc. Natl. Acad. Sci. USA 80:402-406 (1983); Theriault and Roy, Gene 19:355-359 (1982); PvuII: Blumenthal et al., J. Bacteriol. 164:501-509 (1985); Tsp45I: Wayne et al. Gene 202:83-88 (1997)).
A third approach is to select for active expression of methylase genes (methylase selection) (U.S. Pat. No. 5,200,333 and BsuRI: Kiss et al., Nucl. Acids. Res. 13:6403-6421 (1985)). Since R-M genes are often closely linked together, both genes can often be cloned simultaneously. This selection does not always yield a complete restriction system however, but instead yields only the methylase gene (BspRI: Szomolanyi et al., Gene 10:219-225 (1980); BcnI: Janulaitis et al., Gene 20:197-204 (1982); BsuRI: Kiss and Baldauf, Gene 21:111-119 (1983); and MspI: Walder et al., J. Biol. Chem. 258:1235-1241 (1983)).
A more recent method, the xe2x80x9cendo-blue methodxe2x80x9d, has been described for direct cloning of restriction endonuclease genes in E. coli based on the indicator strain of E. coli containing the dinD: :lacZ fusion (Fomenkov et al., U.S. Pat. No. 5,498,535, (1996); Fomenkov et al., Nucl. Acids Res. 22:2399-2403 (1994)). This method utilizes the E. coli SOS response signals following DNA damages caused by restriction endonucleases or non-specific nucleases. A number of thermostable nuclease genes (TaqI, Tth111I, BsoBI, Tf nuclease) have been cloned by this method (U.S. Pat. No. 5,498,535).
Because purified restriction endonucleases, and to a lesser extent, modification methylases, are useful tools for creating recombinant molecules in the laboratory, there is a commercial incentive to obtain bacterial strains through recombinant DNA techniques that produce large quantities of restriction enzymes. Such overexpression strains should also simplify the task of enzyme purification.
The present invention relates to a method for cloning the BpmI restriction endonuclease from Bacillus pumilus into E.coli by methylase selection and inverse PCR amplification of the adjacent DNA of the BpmI methylase gene.
The present invention relates to recombinant BpmI and methods for producing the same. BpmI restriction endonuclease is found in the strain of Bacillus pumilus (New England Biolabs"" strain collection #711). It recognizes double-stranded DNA sequence 5xe2x80x2 CTGGAG 3xe2x80x2 (or 5xe2x80x2CTCCAG3xe2x80x2) and cleaves 16/14 bases downstream of its recognition sequence (N16/N14) to generate a 2-base 3xe2x80x2 overhanging ends.
By methylase selection, a methylase gene with high homology to amino-methyltransferases (N6-adenine methylases) was found in a DNA library. This gene was named BpmI M1 gene (BpmIM1, 1650 bp), encoding a 549-aa protein with predicted molecular mass of 63,702 daltons. There was one partial open reading frame upstream of BpmIM1 gene that displayed 31% amino acid sequence identity to another restriction enzyme Eco57I with similar recognition sequence (Eco57I recognition sequence: 5xe2x80x2CTGAAG N16/N14; BpmI recognition sequence: 5xe2x80x2 CTGGAG N16/N14; A. Janulaitis et al. Nucl. Acids Res. 20:6051-6056, (1992)).
In order to clone the rest of the BpmIRM gene, inverse PCR was used to amplify the adjacent DNA sequence. After four rounds of inverse PCR reactions, an open reading frame of 3030 bp was found upstream of BpmI M1 methylase gene, which encodes a 1009-aa protein with predicted molecular mass of 116,891 daltons. By amino acid sequence comparison of BpmI endonuclease with all known proteins in GenBank protein database, it was discovered that BpmI endonuclease is a fusion of two distinct elements with a possible structural domains of restriction-methylation-specificity (R-M-S). This domain organization is analogous to the type I restriction-modification system with three distinct subunits R, M, and S. Because BpmI is quite distinct to other type IIs restriction enzymes, it is proposed that BpmI belongs to a subgroup of type II restriction enzymes called type IIf (f stands for fusion of restriction-modification domains).
To generate a premodified expression host, the BpmIM1 gene was amplified in PCR and cloned in E. coli strain ER2566. BpmI M1 methylase also modifies XhoI site. XhoI recognition sequence 5xe2x80x2 CTCGAG 3xe2x80x2 is similar to BpmI recognition sequence 5xe2x80x2 CTGGAG 3xe2x80x2 with only one base difference. It was concluded that BpmI M1 methylase may recognize the sequence 5xe2x80x2 CTNNAG 3xe2x80x2 and possibly modify the adenine base to create N6-adenine in the symmetric sequence.
The expression of 3030-bp BpmIRM gene was quite difficult because of the large size of the PCR porduct. The BpmIRM gene was first amplified by Taq DNA polymerase and cloned into the premodified host, but no BpmI activity was detected. To improve the fidelity of PCR reaction, Deep Vent DNA polymerase was used in PCR. Among 18 clones with the insert, only one clone (#4) displayed partial BpmI activity. This clone was sequenced and confirmed to contain wild type sequence.